Hematopoietic stem cell transplantation (HSCT) is increasingly used in cancer therapy and has been shown to provide significant graft-versus-tumor effects for several cancers. However, significant issues limit the efficacy of HSCT including relapse for the tumor, graft rejection, graft-versus-host disease (GVHD) and a profound period of immune deficiency leaving the patient highly susceptible to opportunistic infections. Natural killer (NK) cells represent critical components of the innate immune response and are being increasingly used as a therapeutic arm in HSCT. However, the increasing complexity of NK cells and their regulation along with the relatively sparse knowledge on NK cell development/recovery after HSCT seriously hampers clinical application of NK cells as an immunotherapeutic approach. NK subsets exist and may differ markedly in their function due to differential licensing. We will build on exciting preliminary data demonstrating that mouse NK cell subsets have markedly opposing and differential effects on HSCT outcome using several preclinical models assessing effects on viral resistance, tumor relapse and donor engraftment/GVHD after congenic or allogeneic HSCT. To do this we propose 3 SPECIFIC AIMS: Specific Aim 1 will build on our data demonstrating that Ly49G2 represents a global activation/development marker of NK cells as it is predominant after HSCT or with general activation and is independent of MHC. This aim will examine the mechanisms underlying the expansion of this and other subsets and determine their functional roles using resistance to mouse cytomegalovirus (MCMV) following congenic HSCT. As preliminary data indicate that NK cell licensing with Ly49A+, Ly49G2+, and Ly49C/I+ subsets can indeed be observed post-HSCT and not in resting mice with regard to viral resistance, we hypothesize that the environment post-HSCT represents a unique means to understand NK cell subset interactions and that the Ly49 family is diverse with regard to function/licensing. Specific Aim 2 will build on our exciting preliminary data demonstrating that host NK cell subsets appear capable of regulating each other consistent with licensing and performing helper or suppressor functions with regard to donor hematopoietic engraftment after allogeneic HSCT. This will characterize these subsets which appear to behave as licensed or unlicensed and seek to expand on their beneficial effects in vivo in allogeneic HSCT. This aim will also determine long-term effects on outcome after HSCT including myeloid and lymphoid reconstitution and GVHD. Specific Aim 3 will build on the data from the previous aims and new data to determine the mechanisms underlying the effects of donor transferred NK cell helper or suppressor/effector subsets with regard to anti-tumor effects after congenic or allogeneic HSCT. This aim will seek to augment these effects with administration of immunomodulating agents (IL-15 and neutralization of TGF-2). Finally, we will determine the effects of the adoptive NK cell therapy using subsets against cancer stem cell (CSC) populations which may represent critical targets for NK cell therapy (using subsets). These aims will not only aid in the characterization of mouse NK cell subsets with regard to function but will also help in developing means to clinically exploit human NK cells or their subsets therapeutically, particularly in the context of HSCT and cancer as human subsets become better defined.